Metabolic evolution of Escherichia coli strains that produce organic acids

ABSTRACT

This invention relates to the metabolic evolution of a microbial organism previously optimized for producing an organic acid in commercially significant quantities under fermentative conditions using a hexose sugar as sole source of carbon in a minimal mineral medium. As a result of this metabolic evolution, the microbial organism acquires the ability to use pentose sugars derived from cellulosic materials for its growth while retaining the original growth kinetics, the rate of organic acid production and the ability to use hexose sugars as a source of carbon. This invention also discloses the genetic change in the microorganism that confers the ability to use both the hexose and pentose sugars simultaneously in the production of commercially significant quantities of organic acids.

GOVERNMENT SUPPORT

This invention was made with United States government support under acontract awarded from the US Department of Energy under Award NumberDE-EE0002878/001. The United States government has certain rights in theinvention.

CROSS-REFERENCE TO RELATED APPLICATIONS

The application is a divisional application of U.S. application Ser. No.13/394,176, filed on Mar. 5, 2012 which is the U.S. national stageapplication of International Patent Application No. PCT/US2010/057111filed on Nov. 17, 2010, which claims the priority of the U.S.Provisional Application Ser. No. 61/281,483, filed on Nov. 18, 2009.

The Sequence Listing for this application is labeled “Seq-List.txt”which was created on Nov. 16, 2010 and is 240 KB. The entire content ofthe sequence listing is incorporated herein by reference in itsentirety.

BACKGROUND OF THE INVENTION

A 2004 U.S. Department of Energy report entitled “Top value addedchemicals from biomass” has identified twelve building block chemicalsthat can be produced from renewable feedstocks. The twelve sugar-basedbuilding blocks are 1,4-diacids (succinic, fumaric and maleic),2,5-furan dicarboxylic acid, 3-hydroxy propionic acid, aspartic acid,glucaric acid, glutamic acid, itaconic acid, levulinic acid,3-hydroxybutyrolactone, glycerol, sorbitol, and xylitol/arabinitol.

Building block chemicals are molecules with multiple functional groupsthat possess the potential to be transformed into new families of usefulmolecules. These twelve building blocks can be subsequently converted toa number of high-value bio-based chemicals or materials.

Many natural metabolites derived from biological fermentative processessuch as dicarboxylic acids, amino acids, and diols have functionalgroups that are suitable for polymerization and chemical synthesis ofpolymers. In the recent years, the efficiency of microorganisms forproducing monomeric chemical compounds suitable for industrial usage hasbeen significantly increased through genetic manipulations. However, thecost of producing industrial chemicals through biological fermentativeprocess is still very high. At present the biological fermentativeprocesses for the production of industrial chemicals use purifiedcarbohydrates such as glucose and corn starch as the source of carbonand thereby add cost to the fermentative process for producingindustrial chemicals.

The cost of the fermentation process for producing industrial chemicalscan be dramatically reduced by using lignocellulosic biomass as thesource of carbon in the fermentation process. Lignocellulosic biomasscan be obtained from a number of sources including agriculturalresidues, food processing wastes, wood, and wastes from the paper andpulp industry. Biomass consists of roughly 40-50% of hexose sugars(sugars with six carbon atoms) and 10-30% of pentose sugars (sugars withfive carbon atoms). The hexose sugars are known in the art as C6 sugars.The pentose sugars are known in the art as C5 sugars. When hydrolyzed,the lignocellulosic materials yield a mixture of sugars that includesglucose, xylose, arabinose, mannose and galactose. However, a number offermentation processes for the production of industrial chemicals havebeen developed with pure glucose as a source of carbon for their growthand metabolism. For example, the E. coli strain described in U.S. Pat.No. 7,223,567 uses a rich medium supplemented with glucose as the sourceof carbon. The E. coli strain KJ122 useful for the production ofsuccinic acid described by Jantama et al (2008a; 2008b) and in thepublished PCT Patent Applications Nos. WO/2008/021141A2 andWO2010/115067A2 can grow on a minimal medium but still requires glucoseor another sugar as the source of carbon. It would be ideal if theseorganisms with the ability to produce industrial chemicals at highefficiency could be grown in a mixture of sugars derived from hydrolysisof lignocellulose. The inventors have discovered a method to enable themicroorganisms already optimized to produce a specialty industrialchemical to use a mixture of C5 and C6 sugars derived from hydrolysis oflignocellulosic feedstock.

The ability of the microorganism to use multiple sugars simultaneouslyis limited by the existence of certain biochemical regulatory systems.These biochemical regulatory systems within the microbial cells have agenetic basis. Efforts have been made to overcome these regulatorysystems through genetic manipulations.

In many cases industrial microorganisms are grown in a medium containingglucose or sucrose as the source of carbon. The presence of glucose inthe growth medium suppresses the use of other sugars in E. coli andother species of industrial microorganisms. The consumption of othersugars such as xylose, a pentose sugar, by these microorganisms isinitiated only after glucose in the growth medium has been fullyconsumed. This phenomenon related to carbon utilization in industrialmicroorganisms is referred to as catabolite repression or diauxicgrowth. A method to make the microorganisms co-utilize the differentsugars such as C5 and C6 sugars through a relief of cataboliterepression during the production of industrial chemicals in a commercialscale would be critical to lowering the cost of industrial chemicalsproduced by fermentation.

Microorganisms take up sugars through a set of transporter proteinslocated in the cytoplasmic membrane. The microbial sugar transportersfall within three major categories. The largest group of sugartransporters in bacteria is known as ATP binding cassette (ABC)transporters. As the name implies, the ABC transporters require amolecule of ATP for every molecule of sugar transported into thebacterial cell. XylFGH is an ABC transporter for the transport ofxylose, a pentose sugar, into the cell. AraFGH is an ABC transporter forthe transport of arabinose, yet another pentose sugar.

The second type of bacterial sugar transporters are grouped under MajorFacilitator Super family (MFS). Within the MFS sugar transporters, twodifferent categories of transporter are recognized. MFS includesH⁺-linked symporters, Na⁺-linked symporters-antiporters and uniporters.The uniporters are simple facilitators for the sugar transport and donot require a molecule of ATP for every molecule of sugar transportedinto the cell. The trans-membrane protein Glf in Zymononas mobilis is anexample of a facilitator. The H⁺-symporters require an extracellularproton for every sugar molecule transported into the cell. The GalPprotein in E. coli is a symporter for the transport of galactose, ahexose sugar, into the cell. GalP is a very well characterized symporterwith 12 trans-membrane loops. GalP is also reported to have the abilityto transport glucose across the cell membrane. AraE is a proton-linkedsymporter for the transport of arabinose across the cell membrane.Similarly XylE protein is a proton-linked symporter for the transport ofxylose.

The third sugar transporter primarily responsible for the uptake ofhexose sugars such as glucose is known as thephosphoenolpyruvate:carbohydrate phosphotransferase system (PTS). As away of differentiating the other two sugar transport systems from PTS,the other two sugar transport systems (ABC transporters and members ofMFS transporters) are referred as non-PTS sugar transporters. Transferof the phosphoryl group from phosphoenolpyruvate (PEP) catalyzed by thePTS drives the transport and phosphorylation of glucose and other sugarsand results in the formation of phosphorylated sugars and pyruvic acidinside the cell. PTS generated pyruvic acid is apparently not recycledto PEP under aerobic culture conditions where glucose is the sole sourceof carbon. Rather, pyruvate is oxidized by way of the tricarboxylic acidcycle to carbon dioxide. Thus, for the transport of every singlemolecule of glucose, a molecule of PEP is consumed. In terms of cellularbioenergetics, the transport of sugars through PTS is an energyintensive process. Therefore in cells growing anaerobically, where thereis a need to conserve the phosphoenolpyruvate content within the cellsfor the production of industrially useful chemicals, it is desirable toreplace the PTS with other non-PTS sugar transporters not requiring amolecule of PEP for every molecule of sugar transported into the cell.

The PTS is comprised of two cytoplasmic components named EI and HPr anda membrane-bound component EII. E. coli contains at least 15 differentEII complexes. Each EII component is specific to a sugar type to betransported and contains two hydrophobic integral membrane domains (Cand D) and two hydrophilic domains (A and B). These four domainstogether are responsible for the transport and phosphorylation of thesugar molecules. EI protein transfers the phosphate group from PEP toHPr protein. EII protein transfers the phosphate group fromphosphorylated HPr protein to the sugar molecule.

EI is encoded by the ptsI gene. HPr is encoded by the ptsH gene. Theglucose-specific EII complex of enteric bacteria consists of twodistinct proteins namely, EIIA^(Glc) encoded by the gene crr and themembrane-associated protein EIICB^(Glc) encoded by the gene ptsG. ThePTS mediated sugar transport can be inhibited by means of mutating oneof these genes coding for the proteins associated with PTS. Functionalreplacement of PTS by alternative phosphoenolpyruvate-independent uptakeand phosphorylation activities (non-PTS) has resulted in significantimprovements in product yield from glucose and productivity for severalclasses of metabolites.

With the decrease in the PTS-mediated glucose uptake, other systems forglucose uptake can be activated to assure the continued availability ofglucose within the cell for the production of the industrially usefulchemicals. For example, the glf gene coding for glucose permease, aglucose uniporter, has been shown to substitute for the loss of PTSmediated glucose uptake. Similarly the over expression of galP and glkgenes are reported to enhance the glucose uptake and phosphorylation inthe pts⁻ strain of E. coli. GalP is a symporter for the uptake ofgalactose, a hexose sugar. GalP has been reported to transport glucosein the pts⁻ strain. The significance of GalP mediated glucose uptake isevidenced by the fact that the inactivation of galP gene in the pts⁻mutant is found to prevent growth on glucose (Yi et al., 2003). In theabsence of a PTS, Glk is necessary to achieve the phosphorylation of theglucose molecule before it can enter into glycolysis. The expression ofthe GalP protein in a pts⁻ stain can be achieved either by expressingthe galP gene under a constitutive promoter or by means of relieving therepression of the galP gene expression through mutations in genes codingfor the repressor of the galP gene such as galS and galR.

Besides reducing the energy cost incurred in the transport of sugarsinto the cells, the introduction of a mutations into a gene coding for aprotein associated with PTS is expected to relieve the cataboliterepression which in turn would allow the simultaneous transport andutilization of all the sugars present in the culture medium includingthe pentose and hexose sugars. Hernandez-Montalvo et al (2001) studiedthe utilization of a sugar mixture comprising glucose, arabinose andxylose by an E. coli strain devoid of PTS for the transport of glucose.The pts⁻ mutant was able to uptake sugars by a non-PTS mechanism asrapidly as its wild-type parental strain. In cultures grown in minimalmedium supplemented with glucose-xylose or glucose-arabinose mixtures,glucose repressed arabinose or xylose-utilization in the wild typestrain. Under the same culture conditions, the pts⁻ mutantco-metabolized glucose and arabinose. However, glucose still exerted apartial repressive effect on xylose consumption. In cultures growingwith a triple mixture of glucose-arabinose-xylose, the wild type strainsequentially utilized glucose, arabinose and finally xylose. Incontrast, the pts⁻ strain co-metabolized glucose and arabinose, whereasxylose was utilized after glucose-arabinose depletion. As a result ofglucose-arabinose co-metabolism, the pts⁻ strain consumed the totalamount of sugars contained in the culture medium 16% faster than thewild type strain.

A pts⁻ mutant strain with the capacity to co-metabolize glucose andxylose would cause further increase in the rate of consumption of sugarin the medium leading to an increase in productivity. Thus there is aneed in the art for a microorganism that could co-metabolize glucose andxylose since these two sugars represent the predominant sugars that arepresent in the raw cellulosic hydrolysate. Moreover, it has beenreported that the elimination of the ptsG gene function could decreasethe rate of growth of microorganisms metabolically engineered to produceorganic acids (Sanchez et al., 2005). Therefore there is an additionalneed to achieve the ability to use multiple sugars simultaneouslywithout compromising the growth rate and rate of production ofcommercially important chemicals and chemical intermediates.

The objective of the present invention was to metabolically evolvemicroorganisms capable of producing high levels of industrial chemicalsusing multiple sugars simultaneously without reducing the productivity.The inventors have surprisingly identified a process for makingmicroorganisms that simultaneously consume multiple sugars throughmetabolic evolution. This process of metabolic evolution allows thecells to acquire the ability to use multiple sugars without affectingany of its original characteristics such as rapid growth, and theability to produce specific industrial chemicals at commerciallysignificant quantities.

The inventors have also identified a novel genetic basis for the abilityof the microorganism to use glucose and xylose simultaneously.Whole-genome sequencing was used to identify the genetic modificationthat confers to the microorganisms the ability to use multiple sugarssimultaneously in the production of organic acid.

Prior to the present invention, it would have been doubtful whether theability to utilize both hexose sugars and pentose sugars simultaneouslycould be accomplished through a simple genetic manipulation. The presentinvention related to molecular genetics offers the potential to achievethe ability to metabolize efficiently the entire range ofbiomass-derived sugars. For the first time, the present inventionprovides a genetic approach for achieving simultaneous glucose andxylose uptake that is not obligately coupled to the expenditure ofphosphoenol pyruvate.

BRIEF SUMMARY OF THE INVENTION

We have unexpectedly discovered that the microorganism geneticallymodified and optimized for producing commercially significant quantitiesof organic acids through a fermentative process in a minimal growthmedium containing glucose as the source of carbon can further bemetabolically evolved to use multiple sugars simultaneously as a sourceof carbon while maintaining the original optimized organic acidproduction rate.

It is an objective of the present invention to provide a method forconferring to the microorganisms producing organic acid throughfermentative process the ability to use multiple types of sugarmolecules simultaneously as a source of carbon for their growth andorganic acid production. It is another objective of the presentinvention to provide a fermentation process that produces high yields oforganic acids using raw cellulosic hydrolysate.

A feature of the invention is the utilization of the process ofmetabolic evolution to enable the microorganisms genetically modifiedand optimized for producing organic acids from glucose to acquire theability to use hexose and pentose sugars simultaneously.

In one embodiment of the present invention, an E. coli bacterium capableof producing an organic acid in a medium with glucose as a source ofcarbon is metabolically evolved to use additional types of sugar as asource of carbon while maintaining the same rate of organic acidproduction and retaining the capability to use glucose as a source ofcarbon.

In a preferred embodiment, the present invention provides an E. colibacterium capable producing organic acid in a minimal growth mediumsimultaneously using more than one type of sugar.

In yet another preferred embodiment, the present invention provides anE. coli bacterium capable of producing organic acid in a minimal growthmedium using plant hydrolysate including lignocellulosic hydrolysate asthe source of carbon.

In yet another preferred embodiment of the present invention, an E. colibacterium producing succinic acid using glucose and xylosesimultaneously is provided.

In yet another preferred embodiment of the present invention, an E. colibacterium producing succinic acid in a minimal growth medium using planthydrolysate including lignocellulosic hydrolysate is provided.

The present invention is especially useful for producing highly purifiedorganic acids in a very cost-effective manner through biologicalfermentative process using lignocellulosic materials.

In yet another embodiment, the present invention provides a method formaking a microorganism which uses multiple sugars simultaneously bymeans of mutating the genes coding for non-PTS transporter proteins inaddition to reducing the activity of a gene coding for a proteinassociated with a PTS sugar transporter.

In a more preferred embodiment, a microorganism having PTS sugartransporter with reduced activity and a mutated form of galactosesymporter is provided.

Additional advantages of this invention will become readily apparentfrom the ensuing description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Fermentation profile for KJ122 strain of E. coli in mineralsalts medium supplemented with 8% xylose. Fermentation was carried outfor a total period of 168 hours. The xylose utilization shown in solidcircles started around 96 hours accompanied by an increase in thebacterial cell density measured in terms of an increase in opticaldensity at 550 nm shown in open circles. The increase in succinateconcentration shown in solid squares occurred around 96 hours. Alsoshown in the figure are the changes in the concentration of acetate,pyruvate and malate in the medium during the course of 168 hours offermentation.

FIG. 2. Fermentation profile for KJ122 strain of E. coli adapted tometabolize xylose in mineral medium supplemented with 8% xylose.Fermentation was carried out for a total period of 168 hours. The xyloseutilization shown in solid circles started around 72 hours accompaniedby an increase in the bacterial cell density measured in terms of anincrease in optical density at 550 nm shown in open circles. Theincrease in succinate concentration shown in solid squares occurredaround 72 hours. Also shown in the figure are the changes in theconcentration of acetate, pyruvate and malate in the medium during thecourse of 168 hours of fermentation.

FIG. 3. Fermentation profile for TG400 strain of E. coli in mineralmedium supplemented with 8% xylose. The fermentation profile wasmonitored for a period of 120 hours. The xylose utilization shown insolid circles started around 30 hours accompanied by an increase in thebacterial cell density measured in terms of an increase in opticaldensity at 550 nm shown in open circles. The increase in succinateconcentration shown in solid squares occurred around 30 hours. Alsoshown in the figure are the changes in the concentration of acetate,pyruvate, and malate in the medium during the course of 120 hours offermentation.

FIG. 4. Profile of mixed sugar fermentation by KJ122 and TG400 strainsof E. coli. The fermentation was monitored for a total period of 144hours. The fermentation medium contained both glucose and xylose. Theglucose utilization as shown by a decrease in glucose concentration isshown by open squares. The xylose utilization as shown by a decrease inxylose concentration is shown by solid circles. The increase in thesuccinate concentration in the fermentation medium is shown by solidsquares. The change in the cell density as measured by optical densityat 550 nm during the course of 144 hours of fermentation is shown byopen circles. Also shown in figures are the changes in the concentrationof acetate, pyruvate and malate during the course of 144 hours offermentation.

FIG. 5. Profile of fermentation of detoxified concentrated bagasse C5hydrolysate supplemented with 2.5% (w/v) corn steep liquor by TG400strain of E. coli. The fermentation was carried out for a period of 168hours. The xylose utilization as measured by a decrease in theconcentration of xylose is shown in solid circles. The increase insuccinate concentration in the fermentation medium is shown by solidsquares. Also shown in the figure are the changes in the concentrationof pyruvate, acetate, malate and lactate in the medium during the courseof 168 hours of fermentation.

FIG. 6. Fermentation profile of WG37 strain of E. coli in a mediumcontaining both glucose and xylose. Fermentation was carried out for aperiod of 120 hours. The glucose utilization as measured by a decreasein the glucose concentration in the medium is shown in open squares. Thexylose utilization as measured by a decrease in the xylose concentrationin the medium is shown in solid circles. The change in the bacterialcell density as measured by optical density at 550 nm during the courseof 120 hours of fermentation is shown by open circles. The increase inthe succinate concentration is shown in sold squares. Also shown in thefigure are the changes in the concentration of acetic acid, pyruvic acidand malic acid during the course of 120 hours of fermentation.

FIG. 7. Side-by-side comparison of succinic acid production by TG400,WG37 and KJ122 strains of bacteria in the growth medium containing 4%xylose and 7% glucose. The fermentation was carried out for a period of120 hours. The increase in the concentration of succinic acid in thefermentation medium with TG400 (solid circles), KJ122 (solid triangle),and WG37 (inverted triangle) strains of E. coli was monitored for aperiod of 120 hours.

FIG. 8. Side-by-side comparison of succinic acid production by TG400,WG37 and KJ122 strains of bacteria in the growth medium containing only10% xylose. The fermentation was carried out for a period of 120 hours.The increase in the concentration of succinic acid in the fermentationmedium with TG400 (solid circles), KJ122 (inverted triangle), and WG37(triangle) strains of E. coli was monitored for a period of 120 hours.

FIG. 9. Side-by-side comparison of succinic acid production by TG400,WG37 and KJ122 strains of bacteria in the growth medium containing only10% glucose. The fermentation was carried out for a period of 120 hours.The increase in the concentration of succinic acid in the fermentationmedium with TG400 (solid circles), KJ122 (triangle), and WG37 (invertedtriangle) strains of E. coli was monitored for a period of 120 hours.

FIG. 10. Growth Profile KJ122 and SI014 strains of E. coli in a growthmedium containing xylose as the sole source of carbon. The bacterialgrowth was monitored in terms of optical density at 600 nm for a totalperiod 97 hours. KJ122 strain of (solid circles) showed only a very slowgrowth. On the other hand, SI014 strain (solid squares) showed a fastgrowth within 27 hours followed by a slow decrease in the cell density.

FIG. 11. Profile for xylose utilization and succinate production byKJ122 and SI014 strains of E. coli in a medium containing xylose as thesole source of carbon. The xylose utilization was measured in terms of adecrease in the xylose concentration in the medium for a period of 97hours, Xylose utilization with SI014 strain (solid squares) was muchfaster when compared to the xylose utilization by KJ122 strain (solidcircle). Similarly, the succinic production with SI014 strain (opensquares) was much faster when compared to the succinic acid productionby KJ122 strain (open circles).

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The present invention provides a process for the production of organicacids in commercially significant quantities from the fermentation ofcarbon compounds by recombinant microorganisms. More specifically, thispresent invention provides the microorganisms suitable for theproduction of organic acid through fermentative process. Themicroorganisms of the present invention possess the ability to usemultiple sugars in the fermentative process for the production ofcommercially significant quantities of organic acid.

Disclosed in this present invention are the microorganisms suitable forthe production of succinic acid through fermentative process. Althoughthe present invention provides a process for the production of succinicacid in commercially significant quantities from the carbon compounds bygenetically modified bacterial strains, the teachings of the presentinvention are equally applicable to the industrial production of anumber of other chemicals.

For the purpose of the description of the present invention, thefollowing definitions shall be used.

A number of industrially useful chemicals can be manufactured using thepresent invention. Examples of such chemicals include, but are notlimited to, ethanol, butanols, lactate, succinate, fumarate, malatethreonine, methionine and lysine. Since organic acids can exist both asfree acids and as salts (for example, but not limited to, salts ofsodium, potassium, magnesium, calcium, ammonium, chloride, sulfate,carbonate, bicarbonate, etc), chemical names such as succinic acid,fumaric acid, malic acid, aspartic acid, threonine, methionine, andlysine shall be meant to include both the free acid and any saltthereof. Likewise, any salt, such as succinate, fumarate, malate,aspartate, etc., shall be meant to include the free acid as well.

The present invention combines the technique of specific geneticmodifications with the process of metabolic evolution to obtain strainsshowing high yield, titer and volumetric productivity for succinic acidproduction under anaerobic growth condition in the mineral salt mediumwith a carbohydrate substrate.

As used in the present invention, the term “titer” means the molarconcentration of particular compound in the fermentation broth. Thus inthe fermentation process for the production of succinic acid accordingto the present invention, a succinic acid titer of 100 mM would meanthat the fermentation broth at the time of measurement contained 100mMoles of succinic acid per liter of the fermentation broth.

As used in the present invention, the term “yield” refers to the molesof particular compound produced per mole of the feedstock consumedduring the fermentation process. Thus in the fermentative process forthe production of succinic acid using glucose as the feedstock, the termyield refers to the number of moles of succinic acid produced per moleof glucose consumed.

As used in the present invention, the term “volumetric productivity”refers to the amount of particular compound in grams produced per unitvolume per unit time. Thus a volumetric productivity value of 0.9 gL⁻¹h⁻¹ for succinic acid would mean that 0.9 gram succinic acid isaccumulated in one liter of fermentation broth during an hour of growth.

As used in the present invention, the term “gene” includes the openreading frame of the gene as well as the upstream and downstreamregulatory sequences. The upstream regulatory region is also referred asthe promoter region of the gene. The downstream regulatory region isalso referred as the terminator sequence region.

The phrase “functionally similar” means broadly any wild type or mutatedDNA sequence, gene, enzyme, protein, from any organism, that has abiological function that is equivalent or similar to any wild type ormutated DNA sequence, gene, enzyme, protein that is found in the same ora different organism by the methods disclosed herein. Functionallysimilarity need not require sequence homology. Allele is one of two ormore forms of DNA sequence of a particular gene. Each gene has differentalleles. A gene without any mutation is referred as a wild type allelewhen compared to a corresponding gene that has a mutation.

A homolog is a gene related to a second gene by descent from a commonancestral DNA sequence. The term, homolog, may apply to the relationshipbetween genes separated by the event of speciation or to therelationship between genes separated by the event of geneticduplication. Orthologs are genes in different species that evolved froma common ancestral gene by speciation. Normally, orthologs retain thesame function in the course of evolution. Identification of orthologs iscritical for reliable prediction of gene function in newly sequencedgenomes. Speciation is the origin of a new species capable of making aliving in a new way from the species from which it arose. As part ofthis process it has also acquired some barrier to genetic exchange withthe parent species. Paralogs are genes related by duplication within agenome. Orthologs retain the same function in the course of evolution,whereas paralogs evolve new function, even if these are related to theoriginal one.

A gene or protein with “altered activity” is broadly defined as gene orprotein that produces a measurable difference in a measurable propertywhen compared to the relevant wild type gene or protein. The alteredactivity could manifest itself in a general way by increasing ordecreasing the growth rate or efficiency of succinate production of thestrain containing the altered gene or protein. Other measurableproperties include, but are not limited to enzyme activity, substratespecificity of an enzyme, kinetic parameters of an enzyme such asaffinity for a substrate or rate, stability of an enzyme, regulatoryproperties of an enzyme, gene expression level, regulation of geneexpression under various conditions, etc.

As used in the present invention, the term mutation refers to geneticmodifications done to the gene including the open reading frame,upstream regulatory region and downstream regulatory region. The genemutations result either in an up regulation or a down regulation orcomplete inhibition of the transcription of the open reading frame ofthe gene. The gene mutations are achieved either by deleting the entirecoding region of the gene or a portion of the coding nucleotide sequenceor by introducing a frame shift mutation, a missense mutation, andinsertion, or by introducing a stop codon or combinations thereof.Mutations may occur in the structural genes coding for the proteinsdirectly involved in the biological functions such as enzyme reactionsor transport of the organic molecules across the cell membrane.Alternately, mutations may occur in the regulatory genes coding for theproteins which control the expression of the genes coding for theproteins directly involved in the biological functions. The proteinswhich control the expression of the other genes are referred asregulatory proteins and the genes coding for these regulatory proteinsare referred as regulatory genes.

“Mutation” shall also include any change in a DNA sequence relative tothat of the relevant wild type organism. For example, a mutation foundin strain KJ122 is any change in a DNA sequence that can be found whenthe DNA sequence of the mutated region is compared to that of the parentwild type strain, E. coli C, also known as ATCC 8739. A mutation can bean insertion of additional DNA of any number of base pairs or a deletionof DNA of any number of base pairs. A particular type of insertionmutation is a gene duplication. A gene can be duplicated by aspontaneous mutational event, in which the second copy of the gene canbe located adjacent to the original copy, or a gene can be duplicated bygenetic engineering, in which the second copy of the gene can be locatedat a site in the genome that is distant from the original copy. Amutation can be a change from one base type to another base type, forexample a change from an adenine to a guanine base. In the vernacular ofgenetics, a mutation can be a missense (which changes the amino acidcoded for by a codon), a nonsense (which changes a codon into stopcodon), a frameshift (which is an insertion or deletion of a number ofbases that is not a multiple of three and which changes the readingframe and alters the amino acid sequence that is encoded downstream fromthe mutation, and often introduces a stop codon downstream from themutation), or an inversion (which results from a DNA sequence beingswitched in polarity but not deleted).

A “null mutation” is a mutation that confers a phenotype that issubstantially identical to that of a deletion of an entire open readingframe of the relevant gene, or that removes all measurable activity ofthe relevant gene.

A “mutant” is a microorganism whose genome contains one or moremutations.

As used in this invention, the term “exogenous” is intended to mean thata molecule or an activity derived from outside of a cell is introducedinto the host microbial organism. In the case an exogenous nucleic acidmolecule introduced into the microbial cell, the introduced nucleic acidmay exist as an independent plasmid or may get integrated into the hostchromosomal DNA. The exogenous nucleic acid coding for a protein may beintroduced into the microbial cell in an expressible form with its ownregulatory sequences such as promoter and terminator sequences.Alternatively, the exogenous nucleic acid molecule may get integratedinto the host chromosomal DNA and may be under the control of the hostregulatory sequences.

The term “endogenous” refers to the molecules and activity that arepresent within the host cell. When used in reference to a biosyntheticactivity, the term “exogenous” refers to an activity that is introducedinto the host reference organism. The source can be, for example, ahomologous or heterologous encoding nucleic acid that expresses thereferenced activity following introduction into the host microbialorganism. If the nucleic acid coding for a protein is obtained from thesame species of the microbial organism, it is referred as homologousDNA. If the nucleic acid derived from a different microbial species, itis referred as heterologous DNA. Irrespective of the nature of the DNA,whether it is homologous or heterologous, when introduced into a hostcell, the DNA as well as the activity derived form that introduced DNAis referred as exogenous. Therefore, exogenous expression of an encodingnucleic acid of the invention can utilize either or both heterologousand homologous encoding nucleic acid.

A cell that “utilizes C5 and C6 sugars simultaneously” means a cell thatconsumes at a measurable rate, and without any substantial delay at thebeginning of an inoculation of said cell into a medium, both a C5 sugar,such as xylose, arabinose, ribose, etc., and a C6 sugar, such asglucose, fructose, galactose, etc., when the cell is grown in a mediumthat contains a substantial concentration of both a C5 and a C6 sugar.The medium containing both a C5 and a C6 sugar can be made from purifiedsugars, or it can be derived from a biomass hydrolysate.

A number of microorganisms including Escherichia coli, Citrobactorfreundii, Gluconobacter oxydans, Gluconobacter asaii, Achromobacterdelmarvae, Achromobacter viscosus, Achromobacter lacticum, Agrobacteriumtumefaciens, Agrobacterium radiobacter, Alcaligenes faecalis,Arthrobacter citreus, Arthrobacter tumescens, Arthrobacter paraffineus,Arthrobacter hydrocarboglutamicus, Arthrobacter oxydans, Aureobacteriumsaperdae, Azotobacter indicus, Brevibacterium ammoniagenes, divaricatum,Brevibacterium lactofermentum, Brevibacterium flavum, Brevibacteriumglobosum, Brevibacterium fuscum, Brevibacterium ketoglutamicum,Brevibacterium helcolum, Brevibacterium pusillum, Brevibacteriumtestaceum, Brevibacterium roseum, Brevibacterium immariophilium,Brevibacterium linens, Brevibacterium protopharmiae, Corynebacteriumacetophilum, Corynebacterium glutamicum, Corynebacterium callunae,Corynebacterium acetoacidophilum, Corynebacterium acetoglutamicum,Enterobacter aerogenes, Erwinia amylovora, Erwinia carotovora, Erwiniaherbicola, Erwinia chrysanthemi, Flavobacterium peregrinum,Flavobacterium fucatum, Flavobacterium aurantinum, Flavobacteriumrhenanum, Flavobacterium sewanense, Flavobacterium breve, Flavobacteriummeningosepticum, Micrococcus sp. CCM825, Morganella morganii, Nocardiaopaca, Nocardia rugosa, Planococcus eucinatus, Proteus rettgeri,Propionibacterium shermanii, Pseudomonas synxantha, Pseudomonasazotoformans, Pseudomonas fluorescens, Pseudomonas ovalis, Pseudomonasstutzeri, Pseudomonas acidovolans, Pseudomonas mucidolens, Pseudomonastestosterone, Pseudomonas aeruginosa, Rhodococcus etythropolis,Rhodococcus rhodochrous, Rhodococcus sp. ATCC 15592, Rhodococcus sp.ATCC 19070, Sporosarcina ureae, Staphylococcus aureus, Vibriometschnikovii, Vibrio tyrogenes, Actinomadura madurae, Actinomycesviolaceochromogenes, Kitasatosporia parulosa, Streptomyces coelicolor,Streptomyces flavelus, Streptomyces griseolus, Streptomyces lividans,Streptomyces olivaceus, Streptomyces tanashiensis, Streptomycesvirginiae, Streptomyces antibioticus, Streptomyces cacaoi, Streptomyceslavendulae, Streptomyces viridochromogenes, Aeromonas salmonicida,Bacillus pumilus, Bacillus circulans, Bacillus thiaminolyticus, Bacilluslicheniformis, Bacillus subtilis, Bacillus amyloliquifaciens, Bacilluscoagulans, Escherichia freundii, Microbacterium ammoniaphilum, Serratiamarcescens, Salmonella typhimurium, Salmonella schottmulleri, Klebsiellaoxytoca, Klebsiella pneumonia or Xanthomonas citri are suitable for thepresent invention. The recombinant microorganisms most suitable for thispresent invention are derived preferably from the Enterobacteriaceaefamily. The preferred microorganisms are selected form the generaEscherichia, Erwinia, Providencia, Klebsiella, Citrobacter and Serratia.The genus Escherichia is most preferred. Within the genus Escherichia,the species Escherichia coli is particularly preferred.

E. coli strains capable of producing organic acids in significantquantities are well known in the art. For example, the U.S. PatentApplication Publication No. 2009/0148914 provides strains of E. coli asa biocatalyst for the production of chemically pure acetate and/orpyruvate. The U.S. Pat. No. 7,629,162 provides derivatives of E. coliK011 strain constructed for the production of lactic acid. InternationalPatent Applications published under the Patent Cooperation Treaty Nos.WO 2008/115958 and WO 2010/115067 provide microorganism engineered toproduce succinate and malate in a minimal mineral salt medium containingglucose as a source of carbon in a pH-controlled batch fermentation.

The wild type E. coli strains obtained from culture collections such asthe ATCC (American Type Culture Collection) can be geneticallyengineered and subsequently metabolically evolved to obtain a strainwith an enhanced ability to produce one more organic acid incommercially significant amounts.

The term “genetically engineered” or “genetic engineering” as usedherein refers to the practice of altering the expression of one or moreenzymes in the microorganisms through manipulating the genomic DNA or aplasmid of the microorganism. The genomic manipulations involve eitheraltering, adding or removing specific DNA sequences from the genomicDNA. The genetic manipulations also involve the insertion of a foreignDNA sequence into the genomic DNA sequence of the microorganism. In themost preferred embodiments of the present invention, the geneticmanipulations are accomplished by means of removing specific DNAsequences from the genomic DNA of the microorganisms without introducingany foreign DNA. Certain genetic manipulations necessary to inactivatethe expression of a gene coding for a particular protein productrequires an insertion of a foreign DNA sequence into the genome of themicroorganism. In the most preferred embodiment of the presentinvention, the introduced exogenous DNA sequences are ultimately removedfrom the genomic DNA of the microorganism so that the microorganism atthe end of the genetic engineering process would have no exogenous DNAin its original genomic DNA. Various techniques necessary foraccomplishing the objectives of the preferred embodiment of the presentinvention have been described in detail in Jantama et al (Biotechnologyand Bioengineering 99: 1140-1153 and Biotechnology and Bioengineering101: 881-893). The published U.S. Pat. No. 7,629,162 and U.S. PatentApplication 2009/0148914 and the International patent applicationspublished under the Patent Cooperation Treaty with InternationalPublication Numbers WO 2008/115958 and WO 2010/115067 also describe thegenetic engineering techniques useful in practicing various embodimentsof this present invention. These scientific publications as well aspatent documents are herein incorporated by reference for the purpose ofproviding the details for genetic engineering techniques useful for thepresent invention.

The microorganisms suitable for the practice of present invention can begrown aerobically (in the presence of oxygen) or anaerobically (in thecomplete absence of oxygen) or micro aerobically (with a low rate ofoxygen supply). Alternatively, the microorganisms suitable for thepresent invention can be grown in a dual-phase growth regime, whereinthe microorganism is initially grown in aerobic growth condition toreach a certain level of cell growth before transferring it to anaerobicgrowth condition to achieve the production of desired organic acids incommercially significant quantities. In order to make the microorganismto produce a particular organic acid, various enzymes involved in anumber of microbial metabolic pathways including glycolytic pathway,tricarboxylic acid cycle (also called Krebs cycle or TCA cycle) andglyoxylate stunt can be manipulated by a variety of genetic engineeringtechniques described in the scientific and patent literature cited andincorporated by references in the paragraph above. All of thosereferences are incorporated into this patent application by reference.The details about various microbial metabolic pathways can be found inthe standard biochemistry text books such as Principles of Biochemistry,by Lehninger and Biochemistry by Lubert Stryer.

Depending on the type of organic acid preferred, the metabolic pathwaysare genetically engineered so that a microorganism produces a particularorganic acid of our choice. The microorganisms are capable ofsynthesizing a number of organic acids including lactic acid, aceticacid, and succinic acid. The list of the enzymes that are active in themicrobial fermentative pathway which can be manipulated using the knowngenetic engineering techniques includes, but is not limited to,isocitrate synthetase (aceA), malate synthase (aceB), the glyoxylateshunt operon (aceBAK), acetate kinase-phosphotransacetylase (ackA-pta);aconitase hydratase 1 and 2 (acnA and acnB); acetyl-CoA synthetase(acs); alcohol dehydrogenase (adhE); citrate synthase (citZ); fumaratereductase (frd); lactate dehydrogenases (ldh); malate dehydrogenase(mdh); aceBAK operon repressor (iclR); phosphoenol pyruvate carboxylase(pepC); pyruvate formate lyase (pfl); pyruvate oxidase (poxB); andpyruvate carboxylase (pyc). Besides these genes directly involved in theglycolysis, tricarboxylic acid cycle and glyoxylate stunt of microbialmetabolic pathways, genetic manipulation of the genes involved in theuptake of carbon compounds useful as a source of energy for thesynthesis of organic acid can also be manipulated either to enhance thecarbon uptake or to enhance the efficiency of energy utilization inorganic acid production. For example a decrease in the glucose uptake bya phosphotransferase system (PTS) could help in reducing the energyspent on glucose uptake into the microbial cell. The energy conserved bymanipulating the PTS can be channeled to improve the efficiency oforganic acid production. The phosphotransferase system genes ptsH andptsG can be manipulated to conserve the energy in glucose uptake andthereby improve the efficiency of organic acid production bymicroorganism. Thus by mining the data available in the area ofmicrobial metabolic pathways, one can delete a set of genes so as toblock most of the metabolic pathways and channel the carbon flow to theproduction of a particular organic acid.

Besides the central metabolic pathways and the sugar uptake mechanisms,the carboxylating enzymes within the bacterial cells can also bemanipulated to improve the fermentative production of organic acid. Therole of carboxylating enzymes in the fermentative production is now wellestablished. At least four different types of carboxylating enzymes areknown to be functional within bacterial cells. The phosphoenol pyruvatecarboxylase (PEPcase or PPC) carboxylates phosphoenol pyruvate leadingto the formation of oxaloacetic acid. The malic enzymes carboxylatepyruvic acid leading to the formation of malic acid and requires reducedcofactors such as NADH or NADPH. The third carboxylating enzyme known aspyruvate carboxylase (PYC) carboxylates pyruvic acid to produceoxaloacetic acid. The fourth carboxylating enzyme known asphosphoenolpyruvate carboxykinase (PCK) carboxylates phosphoenolpyruvate to oxaloacetate with the production of one molecule of ATP forevery molecule of oxaloacetate produced from the carboxylation ofphosphoenol pyruvate molecule. Any one of these carboxylating enzyme canalso be manipulated appropriately in the bacterial strains with theability to utilize hexose and pentose sugars simultaneously to improvethe fermentative production of industrially useful chemicals.

The phosphoenolpyruvate carboxykinase (pck) can be geneticallymanipulated to improve the flow of carbon into the tricarboxylic acidcycle. The advantage in improving the activity of pck lies in the factthat this enzyme while carboxylating phosphoenol pyruvate tooxaloacetate, results in the production of a molecule of ATP for everymolecule of oxaloacetate produced. An increase in the ATP yield wouldincrease the growth rate of the cells.

The recruitment of the native pck for fermentative succinate productioncan be achieved by any mutation that positively affects thetranscription of the pck gene. An increase in the level of PCK activitycan be achieved by means of expressing the pck gene in a multicopyplasmid with a native promoter or any other promoter sequence which isknown to increase the gene's expression. Another way to increase theexpression of the pck gene within the cell is to integrate additionalcopies of the pck gene using transposons. In another embodiment of thepresent invention, the native promoter of the pck gene can be replacedby some other promoter elements known to enhance the level of activity.An increased expression of pck gene can also be achieved either bymutation in the promoter region of the gene or by genetic manipulationof the regulatory elements that are known to interact with the promoterregion of the pck gene. The gene coding for a regulator protein of thepck gene can be mutated or deleted or overexpressed in some way in orderto increase the expression of pck gene. A single point mutation (G to Atransition at position—64 relative to the ATG start codon of pck gene)could increase the transcription of the pck gene accompanied by acorresponding increase in the phosphoenol pyruvate carboxykinase enzymeactivity. A similar increase in the pck gene expression can also beachieved by genetically manipulating the genes coding for the proteinsknown to regulate the expression of pck gene.

The production of the organic acid by the genetically engineeredmicroorganism can be confirmed and quantified by using appropriatetechniques well known in the art. For example, HPLC techniques can beused to measure the quantity of the organic acid produced by theselected clone. The HPLC technology is also helpful in determining thepurity of the organic acid produced by the selected clones.

The microbial organism of the present invention can be grown in a numberof different culture media well known in the field of microbiology. Forexample, the wild type and mutant strains of E. coli are grown inLuria-Bertani (LB) medium containing 1% (w/v) tryptone, 0.5% (w/v) yeastextract, and 0.5% (w/v) NaCl. For the commercial production of theorganic acid using fermentative process involving genetically modifiedmicroorganism as biocatalyst, a minimal mineral salt medium supplementedwith a carbon source is preferred. The use of a minimal mineral saltmedium as opposed to a rich medium like LB medium reduces the cost forthe production of organic acids in a commercial scale. The minimalmineral mediums suitable for the present invention include NBS medium(Causey et al., 2007) and AM1 medium (Martinez et al., 2007). The NBSmedium contains 1 mM betaine, 25.72 mM KH₂PO₄, 28.71 mM K₂HPO₄, 26.50 mM(NH₄)₂HPO₄, 1 mM MgSO₄.7H₂O, 0.1 mM CaCl₂.2H₂O, 0.15 mM Thiamine HCl,5.92 μM FeCl₃.6H₂O, 0.84 μM CoCl₂.6H2O, 0.59 μM CuCl₂.2H₂O, 1.47 μMZnCl₂, 0.83 μM Na₂MoO₄.2H₂O, and 0.81 μM H₃BO₃. The AM1 medium contains1 mM betaine, 19.92 mM (NH₄)₂HPO₄, 7.56 mM NH₄H₂PO₄, 10.5 mM MgSO₄.7H2O,1.0 mM Betaine-KCl, 8.88 μM FeCl₃.6H₂O, 1.26 μM CoCl₂.6H₂O, 0.88 μMCuCl₂.2H₂O, 2.20 μM ZnCl₂, 1.24 μM Na₂MoO₄.2H₂O, 1.21 μM H₃BO₃ and 2.50μM MnCl₂.4H₂O.

Since the accumulation of organic acids in the growth medium tends todecrease the pH of the medium, it is necessary to add appropriateneutralizing agents as required to the culture medium. The pH of theculture vessel can be continuously monitored using a pH probe, andappropriate base can be added to maintain the pH of the growth mediumaround neutral pH. The bases suitable for maintaining the pH of themicrobial culture includes, NaOH, KOH, NH₄HCO₃, Na₂CO₃, NaHCO₃, K2CO₃and (NH₄)₂CO₃. The bases suitable for this purpose can be used alone orin combination.

The mineral medium for microbial growth is supplemented with a carbonsource. The carbon sources useful in the present invention include butare not limited to pentose sugars like xylose, and hexose sugars likeglucose, fructose, and galactose. The carbon source can also besatisfied by providing a combination of different sugars such as acombination of glucose and xylose. The carbon source can also be derivedfrom a hydrolysis of starch or lignocellulose. The hydrolysis of complexcarbohydrates such as starch and lignocelluloses can be achieved eitherby using thermo-chemical conversion processes or enzymatic methods wellknown in the art. The preferred carbon source for the industrialproduction of organic acid using microbial fermentation islignocellulosic hydrolysate derived from the hydrolysis of agriculturalor forestry wastes. The lignocellulosic hydrolysate may further befractionated to yield a hexose-enriched and a pentose-enriched fractionand those fractions can serve as the source of carbon for the commercialproduction of the organic acids using microbial fermentation process.The lignocellulosic hydrolysate can further be detoxified to removecertain chemicals such as furfural which are found to be toxic to anumber of microbial organisms above certain concentrations.

The microbial strains obtained from genetic engineering have theexpected genotype for the production of organic acids. However, theirgrowth rate in the minimal mineral salt medium or the their ability toproduce specific organic acid at the required rate or their ability totolerate certain chemicals in the carbon source derived fromlignocellulosic hydrolysate may not be suitable for using thesegenetically modified microorganism as a biocatalyst for the commercialproduction of organic acid through large scale fermentation process.Genetically modified microbial strains obtained from gene deletions aresubsequently selected for the best representative clone via metabolicadaptation or evolution. During the metabolic evolution, the selectedculture is repeatedly transferred into fresh minimal medium for a periodof time to achieve a clone in which one or more spontaneous mutationsthat occurred during selection results in a phenotype that exhibits fastcell growth, rapid consumption of different carbon sources, ability touse multiple sugars simultaneously, ability to tolerate toxic chemicalsin the carbon source and high production yield and productivity of thedesired organic acid, but low production of other organic acids. Duringthe metabolic evolution, attention is paid to select the clone with thedesirable phenotypes. A microbial organism genetically engineered toproduce a particular organic acid may not have a commercially attractivegrowth rate and consequently may not show the expected yield of thatparticular organic acid. Metabolic evolution can be followed to evolve astrain which shows a significant growth accompanied by an increased ratefor the production of that particular organic acid. A clone resultingfrom the metabolic evolution showing a very good growth rate in mineralmedium supplemented with a carbon source but that has not improved inthe yield of the desired organic acid is not a desirable clone.

The KJ122 strain of E. coli is used in the preferred embodiment of thepresent invention. KJ122 was derived from the wild type E. coli C strainthrough multiple stages involving a combination of both geneticengineering and metabolic evolution. Using genetic engineeringtechniques twelve different genes including lactate hydrogenase (ldhA),alcohol dehydrogenase (adhE), formate transporter (focA), acetate kinase(ackA), pyruvate-formate lyase (pflB), methylglyoxal synthase (msgA),pyruvate oxidase (poxB), propionate kinase with acetate kinase activity(tdcD), α-ketobutyrate formate lyase (tdcE), citrate lyase (citF),aspartate aminotransferase (aspC), and malic enzyme (sfcA) were deletedfrom the chromosomal DNA of the parent E. coli C strain ATCC 8739. Thegenetic manipulations done to E. coli C strain ATCC 8739 leading to theKJ122 strain have been described in detail by Jantama et al (2008a2008b).

During the process of metabolic evolution using selective pressure toforce the organism to acquire a desirable phenotype, two possiblechanges could occur. The organism could simply adapt itself to theselective pressure and show a changed phenotype. Alternatively, theorganism might undergo certain genetic changes under selective pressureand exhibit a changed phenotype permanently. When there was only anadaptation and there is no genetic change, the organism reverts back toits original phenotype once the selection pressure is relieved. Theseorganisms are referred to as “adapted” organisms. The “adapted”microorganisms have to undergo another fresh round of metabolicevolution under selection pressure to show a changed phenotype. On theother hand, when there is an accompanying genetic change, the changedphenotype will continue to exist even when there is no selectionpressure. Metabolic evolution accompanied by a certain genetic change isdesirable. The microorganism acquiring a stable genetic change duringmetabolic evolution can be easily identified by means of growing themicroorganism in the original growth medium without any selectionpressure for some time before transferring it to the fresh medium withthe selection pressure. If these organisms are able to show good growthand the expected phenotype without any lag period, the organism isconsidered to have acquired a changed genotype during metabolicevolution.

The basis of genetic change gained during the metabolic evolution can bedetermined by sequencing the chromosomal DNA of the organism andcomparing the sequence data with that of the parent strain. The genomicsequence data can be obtained by means of following the techniques wellknown in the art. Thus, the parent stain KJ122 obtained from E. colistrain ATCC 8739 can be subjected to metabolic evolution to obtain astrain with a desirable new phenotype. The genome of the metabolicallyevolved new strain along with the parent strain KJ122 can be sequencedand the mutations in the metabolically evolved strain accounting for thechanged phenotype can be identified.

As defined in this invention, the term mutation includes any change inthe nucleotide sequence within the gene. A nucleotide change within agene may be a single nucleotide change within a triplet codon leading tothe replacement of one amino acid residue with another amino acidresidue. Alternately, a nucleotide change within an open reading frameof a gene may involve a deletion of a portion of the open reading frameor the entire open reading frame. A nucleotide change within an openreading frame can also include introduction of a stop codon and as aresult, the open reading frame codes for a truncated protein instead ofa full-length protein. As used in the present invention the termmutation also includes changes in the nucleotide sequences in theupstream or downstream of the open reading frame. The regions upstreamand downstream of an open reading frame contain several regulatorynucleotide sequences and are involved in the expression of the proteincoded by the open reading frame. A mutation occurring in theseregulatory regions can alter the gene expression leading either to anup-regulation or down-regulation of gene function. Another possibilityis a nucleotide insertion or deletion resulting in a frames shiftmutation.

Based on the knowledge gained from the present invention, the geneticmodifications leading to the simultaneous utilization of pentose andhexose sugars can be carried out in any bacterial strain alreadygenetically engineered for the production of one or more industrialchemicals using glucose as the source of carbon. Alternately, thegenetic modification required for the simultaneous utilization of hexoseand pentose sugar can be carried out in any wild type bacterial strainsand the wild type bacterial strain thus modified for simultaneous hexoseand pentose sugar utilization can be subjected to further geneticmodifications to develop a microorganism suitable for the production ofindustrial chemicals in a commercial scale.

EXPERIMENTAL SECTION General Remarks

Strain and Inoculum Preparations:

KJ122 (E. coli C, ΔldhA, ΔadhE, ΔackA, ΔfocA-pflB, ΔmgsA, ΔpoxB, ΔtdcDE,ΔcitF, ΔaspC, ΔsfcA) was used in the present invention. KJ122 wasderived from E. coli C (ATCC 8739) strain through genetic modificationsas described by Jantama et al (2008a; 2008b) and in the InternationalPatent Applications published under Patent Cooperation Treaty withInternational Publication Nos. WO 2008/115958 and WO 2010/115067. Allthese documents are herein incorporated by reference.

E. coli strain KJ122 is capable of fermenting 10% glucose in AM1 mineralmedia to produce 88 g/L succinate, normalized for base addition, in 72hours. AM1 medium contains 2.63 g/L (NH₄)₂HPO₄, 0.87 g/L NH₄H₂PO₄, 1.5mM MgSO₄, 1.0 mM betaine, and 1.5 ml/L trace elements. The traceelements are prepared as a 1000× stock and contained the followingcomponents: 1.6 g/L FeCl₃, 0.2 g/L CoCl₂.6H₂O, 0.1 g/L CuCl₂, 0.2 g/LZnCl₂.4H₂O, 0.2 g/L NaMoO₄, 0.05 g/L H₃BO₃, and 0.33 g/L MnCl₂.4H₂O. ThepH of the fermentation broth is maintained at 7.0 with: 1:4 (6 N KOH: 3M K₂CO₃) (1.2 N KOH, 2.4 M K₂CO₃).

In some experiments, corn steep liquor was added. It is a byproduct fromthe corn wet-milling industry. When compared to the yeast extract andpeptone, it is an inexpensive source of vitamins and trace elements.

Fermentations:

Fermentations were started by streaking on a fresh NBS-2% xylose plate aglycerol stock of E. coli strain genetically engineered to producesuccinic acid and stored in the −80° C. freezer. After 16 hours (37°C.), cells from the plate were scraped off and inoculated directly intothe fermentation vessel. The fermentation vessels have a working volumeof 350 ml. This first fermentation was referred to as the “seed”culture, and was not used to accumulate data. The medium in allfermentations was traditional AM1 medium supplemented with 0.03M KHCO₃,1 mM betaine and 8% xylose (unless otherwise noted) and neutralized witha base consisting of 1.2 N KOH and 2.4 M K₂CO₃. The fermentation vesselswere maintained at a pH of 7.0, 37° C. with 150 rpm stirring. After 24hours, the seed culture was used to inoculate a new culture (whetherbatch experiments or “transfers”) to a starting OD₅₅₀ of 0.05. With theexceptions of the daily transfers, all experiments were conducted intriplicate. The C5/C6 co-fermentation experiment included 4% xylose, 7%glucose, 0.5% arabinose, 0.4% galactose, and 0.3% mannose (pure sugars)and was inoculated from a culture growing on xylose as the sole carbonsource and 0.08% furfural. The C5/C6 co-fermentation was also conductedwith the mixture of 8% xylose and 1% glucose. These experiments wereconducted in triplicate without addition of inhibitors, with 1% acetateor with 0.1% furfural.

Cell Growth:

Cell mass was estimated by measuring the optical density at 550 nm(OD₅₅₀) using a Thermo Electronic Spectronic 20 spectrophotometer.

Organic Acid and Sugar Analysis:

The concentration of various organic acids and sugars were measured byHPLC. Succinic acid and other organic acids present in the fermentationbroth were analyzed on Agilent 1200 HPLC apparatus with BioRad AminexHPX-87H column. BioRad Microguard Cation H⁺ was used as a guard column.The standards for HPLC analysis were prepared in 0.008N sulfuric acid.The HPLC column temperature was maintained at 50° C. Sulfuric acid at0.008N concentration was used as a mobile phase at the flow rate of 0.6ml/min. Quantification of various components was done by measuring theirabsorption at 210 nm.

Metabolic Evolution:

Cells from the pH controlled fermentations were serially transferred at24 hours to encourage metabolic evolution though growth-based selection.The inoculum, approximately 1/100 of the volume of new media, was addeddirectly to pre-warmed, fresh media to a starting OD₅₅₀ of 0.05. Cloneswith improved fermentation characteristics were isolated. The metabolicevolution strategy was applied to improve xylose fermentation.

Preparation of Bagasse:

Sugarcane bagasse is obtained from sugar mills in Florida which is awaste product that is typically used by burning for energy. This wasteproduct is used as the starting material for the preparation ofhemi-cellulose and cellulose fractions using dilute acid pretreatment.

The sugarcane bagasse is dried to a moisture content of about 10% andmilled using a knife mill. The material is treated in steam reactors(Zipperclave & Parr) with dilute sulfuric acid at moderate temperatures.Typical pretreatment conditions for dilute acid pretreatment are 0.1-3%acid concentration, 100-200° C. temperature, and 1-30 minutes residencetime in the reactor. The optimal reactor conditions to achieve maximumxylose yield with minimal sugar degradation are about 0.5% acidconcentration, 160° C., and 10 min resident time in the reactor.

PCR and DNA Sequencing:

A set of two galP specific primers BY38 and BY39 (Table 1), were used toobtain the galP gene from TG400, KJ122, and WG37 strains of E. coli. ThePCR was carried out using the standard protocol using 2phusion HF mastermix kit from New England Biolabs. The PCR products were run on a 0.8%agarose gel to determine the size of the PCR products from each of thesedifferent strains of E. coli. The PCR products were also sequenced usingthe Sanger method by Tufts DNA sequencing core facility in Boston,Mass., USA. The sequence data were analyzed using the Vector NTIsoftware program.

Construction of WG37 Strain of E. coli:

WG37 stain of E. coli was derived from KJ122 strain by deleting theentire coding region of the galP gene. The galP gene was deleted in twosteps involving homologous recombination. In the first stage, the galPgene sequence was replaced by a cassette containing an antibiotic markerand sacB gene sequence. The recombinants were selected on a LB platewith antibiotic. In the second stage, the antibiotic cassette wasremoved from the chromosomal DNA and the recombinants were selected on amedium containing sucrose. The colonies growing on the sucrosecontaining plates are highly enriched for loss of the sacB cassette.

In the construction of the WG37 strain of E. coli, a kan cassette wasamplified by PCR using the primers 51a and 51b (Table 1) and XmnIdigested pGW162 plasmid as a template. The DNA fragment of kan-sacBcassette was introduced into KJ122 strain of E. coli. In the first step,transformants were selected on a LB plate with kanamycin and wereconfirmed by PCR using the primers 49a, 49b (Table 1). This strain wasdesignated as WG35. The galP gene and neighboring 300 bp regions wereamplified using the primers 49a, 49b (Table 1) and cloned into pGEMTeasy vector to produce plasmid pGW180. Diluted preparation of thisplasmid DNA served as a template for inside-out amplification using theprimers 50a, 50b (Table 1). The resulting fragment was self ligated toconstruct plasmid pGW181. In pGW181 (Table 1), the galP gene wasdeleted. The DNA fragment containing the galP deletion was amplified byPCR with the primers 49a, 49b and the plasmid pGW181 as template. ThePCR product was introduced into WG35 and the transformants were selectedon LB plates with 10% sucrose. Resulting clones were tested for loss ofkanamycin resistance. The final galP deletion strain was designated asWG37 and the specific gene deletion was confirmed by PCR using primers49a, and 49b (Table 1).

Example 1 C5 Utilization

Escherichia coli strain KJ122 (E. coli C, ΔldhA, ΔadhE, ΔackA,ΔfocA-pflB, ΔmgsA, ΔpoxB, ΔtdcDE, ΔcitF, ΔaspC, ΔsfcA) was able to growaerobically on glucose, xylose, and arabinose. The objective of thepresent invention was to grow the KJ122 strain of E. colimicroaerobically in a medium containing both hexose and other pentosesugars and to select an organism that is able to use both types ofsugars simultaneously.

Initial screening for C5 utilization was conducted by aerobic growth onNBS mineral medium plates supplemented with 2% of xylose. The plateswere incubated at 37° C. overnight. The colonies appearing on the xyloseplate were streaked on fresh plates for three consecutive times. At theend of the third transfer on solid NBS mineral medium with 2% xylose,the cells from the plate were scraped off and inoculated directly to afermentation flask containing AM1 mineral medium supplemented with 0.03M KHCO₃, 1 mM betaine and 8% xylose. The fermentation medium wasneutralized with a base consisting of 1.2 N KOH and 2.4 M K₂CO₃ tomaintain the pH of 7.0 at 37° C. The culture was stirred with a magneticstir bar operating at the speed of 150 rpm. The liquid culture was grownfor an initial period of 24 hours and used as a seed culture to start anew culture with an initial OD₅₅₀ of 0.05.

This culture in the AM1 mineral medium with xylose exhibited an initial72 hour lag phase during which no growth of KJ122 was noticed. At theend of this initial lag period, KJ122 strain started showing growth.Along with the growth of the bacterial cells as measured by an increasein the OD at 550 nm, there was a decrease in the concentration of xylosein the medium accompanied by a proportional increase in theconcentration of the succinic acid in the growth medium.

At the end of the 216 hour growth period in xylose containing medium, aglycerol stock of this culture was prepared and stored at −80° C. Eitherthe fresh culture at the end of the 216 hour growth period or theglycerol stock of the culture prepared at the end of 216 hour growthperiod was used to inoculate a fresh fermentation vessel with AM1mineral medium supplemented with 8% xylose. Irrespective of the sourceof inoculum, whether it was from a fresh culture or a glycerol stock,the culture in the second fermentation vessel grew without any lagperiod. The succinic acid production also accompanied the bacterialgrowth without any lag period. Thus three rounds of growth on a solidmineral medium with 2% xylose followed by a single growth cycle for 216hour period resulted in the “adapted strain” of KJ122 which is able togrow microaerobically on xylose containing medium.

Example 2 Metabolic Evolution of KJ122

In another embodiment of the present invention, the KJ122 strain wassubjected to metabolic evolution. The KJ122 culture growingmicroaerobically in a liquid AM1 medium supplemented with xylose sugarwas transferred to a fresh liquid AM1 medium containing 8% xylose every24 hours for a period of 2 weeks. At the end of these multipletransfers, the KJ122 strain was transferred to a fresh fermentor withAM1 medium supplemented with 8% xylose. The anaerobic growth rate ofKJ122 in the fermentor as well as the succinic acid production and thekinetics of xylose utilization were monitored. The succinic acidproduction in the fermentor started immediately without any lag periodand also produced higher final titers and this strain is referred as a“metabolically evolved strain.” In our strain collection, thismetabolically evolved strain has been designated as TG400.

In order to determine whether the “adapted strain” of KJ122 and the“metabolically evolved” TG400 strain have any genetic basis for theirchanged phenotype, the following experiments were carried out. Theunmodified KJ122 strain, the “adapted strain of KJ122” and the“metabolically evolved” TG400 strain were streaked onto a plate withfresh mineral medium containing 2% glucose. The resulting colonies werestreaked onto a fresh plate with 2% glucose. This streaking was done ona daily basis for 11 consecutive days. At the end of the eleventh day,the culture was streaked onto an agar plate containing xylose. Thecolonies growing on the xylose plate were streaked again onto a freshxylose containing plate. This was followed by the transfer of thecolonies growing on the second xylose containing plate to a liquidculture. The growth rate, succinic acid production kinetics and the rateof decease in the concentration of the xylose in the culture medium weremonitored.

As the results shown in FIGS. 1, 2, and 3 indicate, the xyloseutilization as monitored by the disappearance of xylose in the growthmedium showed a lag period of 96 hours both in the original KJ122 strainas well as in the “adapted” strain of KJ122. In the case ofmetabolically evolved TG400 strain, most of the xylose in the medium wasconsumed within the first 96 hours. In addition, for TG400, the succinicacid production did not show any lag period. Similarly, the cell growthfor TG400 showed no lag period while the “adapted strain” of KJ122 andthe original KJ122 stain showed an initial lag phase of about 72 hours(FIGS. 1 and 2). These observations clearly establish that themetabolically evolved TG400 strain has acquired a stable genotype forxylose utilization during metabolic evolution and this ability was notlost even when this strain was grown for several generations in theabsence of xylose. On the other hand, the KJ122 strain adapted to growin the medium containing only xylose, loses its ability to use xylose,when grown for several generations in the glucose containing medium inthe absence of xylose. This “adapted strain” of KJ122 grown in theabsence of xylose needed additional 96 hours to adapt itself again touse xylose as a source of carbon. Thus the “adapted strain” of KJ122 didnot acquire any genetic modification during it adaption in the xylosecontaining medium for 96 hours.

TG400 while acquiring an ability to use xylose as a carbon source stillretained its ability to use glucose as the source of carbon (Table 2).

Example 3 C5+C6 Co-Fermentation

In KJ122 under anaerobic growth conditions, the C5 and C6 sugars are notsimultaneously metabolized. The C6 sugars are generally metabolizedfirst, and a lag is exhibited prior to C5 metabolism. Therefore it wasessential to determine the fermentation characteristics of TG400 in thepresence of equal amounts of both C6 and C5 sugars. As shown in FIG. 4,TG400 was able to use glucose and xylose at the same rate and producedsuccinic acid without any lag period. KJ122 was also able to use bothxylose and glucose. However in the KJ122 strain, the xylose utilizationstarted only after a substantial decrease in the glucose concentration.Further as shown in Table 3, TG400 used more xylose on a molar basisthan glucose when compared to the xylose and glucose utilization byKJ122.

Example 4 Fermentation of Detoxified Bagasse Hydrolysate Enriched in C5Sugars

TG400 strain obtained through metabolic evolution was tested for itsability to use xylose derived from a hydrolysis of bagasse. Theconcentrated bagasse hydrolysate was detoxified by means of treating itwith 50 grams of charcoal for every kilogram of bagasse hydrolysate at35° C. for 60 minutes in a rotary shaker at 200 rpm. The activatedcharcoal treated C5 enriched bagasse was pH adjusted, supplemented withAM1 mineral salts, betaine and trace elements and then filtersterilized. Hydrolysate comprised primarily of 8% (w/v) xylose (C5) andapproximately 0.8% glucose (C6), 0.1% galactose (C6), 0.1% mannose (C6)and 0.002% arabinose (C5). The concentrated detoxified C5 sugarsenriched bagasse hydrolysate was further supplemented with 2.5% w/v cornsteep liquor and inoculated with TG400 strain to an initial OD₅₅₀ nm of0.5. As the results shown in FIG. 5 indicate, within 120 hours, all thesugars in the culture medium were consumed and there was a steadyproduction of succinate.

Example 5 Genomic Sequencing of KJ122 and TG400 Strains of Escherichiacoli

The entire genome of the parent strain KJ122 and the TG400 strainderived from KJ122 through metabolic evolution were sequenced using anIllumina Genome Analyzer II at the Tufts University Core Facility inBoston Mass., USA. The Genome Analyzer II is provided by IlluminaSequencing Technology. The genomic data obtained for KJ122 and TG400were compared to each other to identify the genetic changes accompanyingthe metabolic evolution of TG400 from KJ122. A comparative analysis ofTG400 and KJ122 revealed a mutational change in the galP gene of TG400.The galP gene in TG400 showed a point mutation at the nucleotideposition 889 of its open reading frame. The cytosine nucleotide at thisposition was changed to guanosine residue. As a result of thisnucleotide change, the amino residue glycine was changed to aspartate.This mutation in the galP gene is referred as galP*. This mutation wasthe only difference between KJ122 and TG400 strains of bacteria at thenucleotide level.

Example 6 PCR and Sequence Analysis of galP Gene Sequences in TG400 andKJ122

Having established that there is a mutation in the galP gene sequence inthe TG400 strain accounting for its ability to use xylose and glucosesimultaneously, we used PCR techniques to obtain the galP gene fromKJ122 and TG400. The PCR products obtained from TG400 as well as the PCRproduct from KJ122 were sequenced. As the sequence data revealed a pointmutation that changes glycine residue at position 296 to an aspartateresidue (Gly296 to Asp).

Example 7 Effect of galP Gene Deletion

Having established that there is a galP gene mutation in TG400accompanying the ability to use glucose and xylose simultaneously, wedecided to determine whether deletion of the entire galP gene would havethe same phenotypic effect as seen in TG400 with a mutated galP gene. Inthese experiments we used KJ122 of E. coli as the parent strain.

We deleted the galP gene sequence from the KJ122 strain of E. coli toproduce a new strain called WG37. We measured the growth kinetics, sugarutilization pattern and the succinic acid production in KJ122, TG400 andWG37 strains grown anaerobically in a minimal medium containing bothglucose and xylose as the source of carbon. WG37 was able to use bothglucose and xylose simultaneously during the course of 96 hours (FIG.6). Its growth kinetics as well as the sugar utilization patterns weresimilar to that of TG400 which has a mutated form of galP gene. InKJ122, the glucose was completely consumed within 72 hours while thexylose utilization showed an initial lag of 24 hours. Both in TG400 andin WG37, the glucose was not exhausted even after 96 hours of growth anda significant amount of glucose remained in the medium at 96 hours ofgrowth. In addition, both in TG400 and WG37, the xylose utilizationcould be detected as early as 12 hours.

FIGS. 7, 8 and 9 show the side-by-side comparison of kinetics ofsuccinic acid production in all the three strains used in the presentinvention. When grown in the medium containing 4% xylose and 7% glucose,all the strains showed a similar kinetics for succinic acid productionirrespective of whether they had an intact galP gene sequence or not. Inthe medium containing the mixed sugars, TG400 showed a slightly higherrate for succinic acid production (FIG. 7).

In the medium containing xylose as the only source of carbon, the TG400and WG37 strains showed much faster rates for succinic acid productionwhen compared to the rate of succinic acid production by KJ122 strain(FIG. 8).

In the medium containing glucose as the only source of carbon, the twobacterial strains TG400 and WG37 with deletions in galP gene sequenceshowed slower rates for succinic acid production when compared to therate of succinic acid production by KJ122. (FIG. 9).

Example 8 Effect of G297D Point Mutation in galP on Xylose Utilization

In order to examine the effect of point mutation that results in G297D(replacement of glycine residue at position 297 with aspartate residuein the GalP protein) on the growth and succinate production in thefermentation medium containing xylose as the sole source of carbon, theG297D mutation was introduced into the galP gene sequence in the KJ122strain. SI014 (KJ122 ΔgalP::galP*) was created by PCR amplifying themutant galP* gene from TG400 using the primers 17ASPgalP (SEQ ID No. 9)and 18SPgalP (SEQ ID NO. 10) and then recombining within WG35 (KJ122ΔgalP::kan-sacB) which was expressing lambda red recombinase from atemperature conditional plasmid, pKD46. Plasmids pKD46 was then removedby growth at an elevated temperature (Datsenko and Waner, 2000).

KJ122 was obtained off a MacConkey lactose plate. SI014 (KJ122galP*) wastaken from an LB 2% glucose plate. Scrapes from the plates were used toinoculate 25 mls LB 2% glucose. Cultures were grown for 8 hours at 37°C., 150 rpm. Final OD₆₀₀ for these cultures was 0.71 for KJ122 and 0.58for SI014. 5 mls of each LB glucose culture was used to inoculate a 300ml seed fermentor containing AM1 10% glucose medium. These fermentationswere held at pH 7.0, 37° C. for 24 hours. Final OD₆₀₀ for these cultureswas 3.82 for KJ122 and 2.89 for SI014. These cultures were used toinoculate triplicate fermentors containing AM1 10% xylose medium. Targetfinal OD₆₀₀ is 0.1, so for KJ122, 7.85 mls was used and for SI014, 10.38mls was used to inoculate a 300 ml fermentation. Fermentations weremaintained at pH 7.0 and 37 C for 97 hours. Samples were taken daily forOD₆₀₀ and metabolite analysis. All metabolite and growth data wasgraphed and analyzed (2 way ANOVA) using GraphPad Prism.

The growth profile for E. coli strain SI014 which contains a pointmutation in the galP gene that results in 1 amino acid change atposition 297, is shown in FIG. 10. It is very clear by OD₆₀₀ valuesthat, on a medium containing xylose as the sole source of carbon, SI014strain grows more quickly and more densely than KJ122. The only knowndifference between these two strains is the point mutation in the galPgene. FIG. 11 shows the increased consumption of xylose and theconcomitant increase in succinic acid production for strain SI014compared to KJ122.

The applicants' invention has been described in detail above withparticular reference to preferred embodiment. A skilled practitionerfamiliar with the above detailed description can make any modificationwithout departing from the spirit of the claims that follow.

TABLE 1 Nucleotide sequence of the primers used in the present inventionPrimer Primer No. Name Primer sequence SEQ ID No. 1 BY38 5′cagcgtttaatctatgatgatataactc aattattttca 3′ SEQ ID No. 2 BY39 5′ggcgatagggagacgggatgttttc 3′ SEQ ID No. 3 49a 5′ ccgattacaccaaccacaac 3′SEQ ID No. 4 49b 5′ ggcgaatttcatagctttcc 3′ SEQ ID No. 5 50a 5′gaaataggcgctcacgatta 3′ SEQ ID No. 6 50b 5′ aaacgtcattgccttgtttg 3′SEQ ID No. 7 51a 5′ taaccatattggagggcatcatgcctgacgctaaaaaacaggggcggtcaaacaaggcaactag cgcatgcatccattta 3′ SEQ ID No. 8 51b5′ ctgcaagaggtggcttcctccgcgatggga ggaagcttggggagattaatcgtgagcgcctggcgaagaactccagcatga 3′ SEQ ID No. 9 17ASPgalP 5′ acccagcacgttttccatca 3′SEQ ID No. 10 18SPgalP 5′ tgcgttcaaaggccagcctc 3′

TABLE 2 Glucose fermentation by KJ122 and TG 400 strains of E. coli.KJ122 TG400 mM* g/L* mM* g/L* Glucose 573 103 368 66 consumed Succinic742 88 492 58 acid Yield 1.29 (mol/mol) 1.35 (mol/mol) Yield (%) 75% 78%(Theoretical) *All the values provided here are normalized for baseaddition.

TABLE 3 Co-fermentation of C5 and C6 sugars by KJ122 and TG400 stains ofE. coli. TG400 and KJ122 were on glucose containing medium prior toexperiment. TG400*** was actively growing on xylose prior to experiment.The results are average of two fermentations. TG400 KJ122 TG400*** mM*g/L* mM* g/L* mM* g/L* Xylose 195 29 142 221 221 33 consumed Glucose 38970 448 80.8 366 66 consumed Succinic 721 85 633 75 810 96 acidTheoretical 945 112 971 115 948 112 yield Yield (%) 76% 65% 86% *All thevalues provided here are normalized for base addition

TABLE 4 Yields from batch fermentations (8% xylose)—KJ122 vs. TG400 Suc-Pyru- cinate Acetate Malate vate Yield^(b) Time Strain Conditions (g/L)(g/L) (g/L) (g/L) (%) (hrs) KJ122^(c) ^(a, c) 54 4 5 8 61 192 KJ122 ^(a)50 4 1 7 61 120 TG400 ^(a) 70 7. 0 0 76 96 KJ122 C5 + C6^(d) 75 7 5 2 65120 TG400 C5 + C6^(d) 96 10 1 0 86 120 TG400 Hydrolysate^(e) 66 6 1 0 92120 ^(a)Batch fermentations were performed in triplicate in AM1 mineralsalts media supplemented with 0.03 M KHCO₂, 1 mM betaine and 8% xylose,unless otherwise noted; pH was controlled at 7.0 by automatic additionof 1.2 N KOH and 2.4 M K₂CO₃. Titers are normalized for base addition.^(b)Yields are based on metabolized sugar assuming a maximum theoreticalyield of 1.12 g of succinic acid per g of xylose. ^(c)First xylosefermentation (in duplicate) ^(d)Initial sugar concentration: 4% xylose,7% glucose, 0.4% galactose, 0.3% mannose, 0.5% arabinose^(e)Concentrated detoxified C5 hydrolysate from bagasse (see FIG. 5legend). Initial sugar concentration was: 56.6 g/L xylose, 4.5 g/Lglucose, 0.0009 g/L galactose, 2.7 g/L arabinose. All sugars wereconsumed. Initial acetic acid from pretreatment process 4.04 g/L) wassubtracted from final acetate to determine acetic acid produced duringfermentation.

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We claim:
 1. An isolated Escherichia coli bacterial cell comprising amutation in galactose symporter (galP) gene, wherein said bacterial cellcomprises at least one mutation that decreases the activity ofPEP-dependent phosphotransferase system and utilizes C5 and C6 sugarssimultaneously to produce an industrially useful chemical and saidmutation in galP gene is a deletion.
 2. The isolated Escherichia colibacterial cell of claim 1, further comprising at least one mutation thatincreases the activity of at least one non-PTS sugar transporter.
 3. Theisolated Escherichia coli bacterial cell of claim 2, wherein saidnon-PTS sugar transporter is an ATP binding cassette transporter.
 4. Theisolated Escherichia coli bacterial cell of claim 2, wherein saidnon-PTS sugar transporter is a member of major facilitator super family.5. The isolated Escherichia coli bacterial cell of claim 1, furthercomprising at least one mutation that inactivates the expression of oneor more genes involved in a fermentation pathway.
 6. The isolatedEscherichia coli bacterial cell of claim 1, further comprising at leastone mutation that inactivates one or more genes associated with thetricarboxylic acid cycle.
 7. The isolated Escherichia coli bacterialcell of claim 1, further comprising mutation in at least one of thegenes selected from a group consisting of gene coding forphosphoenolpyruvate carboxylase, gene coding for NADH dependent malicenzyme, and gene coding for NADPH dependent malic enzyme.
 8. Theisolated Escherichia coli bacterial strain of claim 1, furthercomprising an exogenous pyruvate carboxylase.
 9. The isolatedEscherichia coli bacterial strain of claim 8, wherein said pyruvatecarboxylase is from Lactobacillus lactis or Sorghum vulgare or Rhizobiumetli.
 10. The isolated Escherichia coli bacterial cell of claim 1,further comprising increased phosphoenol pyruvate carboxykinaseactivity.
 11. The isolated Escherichia coli bacterial cell of claim 10,wherein increased levels of phosphoenol pyruvate carboxykinase activityresult from a mutation in pck gene.
 12. The isolated Escherichia colibacterial cell of claim 11, wherein said mutation is in the promoterregion of said pck gene.
 13. A process for the microbial production ofan organic dicarboxylic acid comprising: a. providing an isolatedEscherichia coli bacterial cell having a mutation in galactose symporter(galP) gene comprises at least one mutation that decreases the activityof PEP-dependent phosphotransferase system and utilizes C5 and C6 sugarssimultaneously to produce an industrially useful chemical dicarboxylicacid and said mutation in galP gene is a deletion; b. culturing thebacterial cell of step (a) in a medium containing both pentose andhexose sugars simultaneously; and c. recovering the organic acid fromthe culture medium.